The fundamental goal of this project is to determine the structural basis of the pathological properties of amyloid fibers involved in currently incurable neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's, and other amyloidoses. The different proteins forming pathological amyloids are folded in hyperstable filaments with a cross-beta peptide conformation, common in fibers of denatured proteins, but very rare in native biological fibers. Microcrystals of amyloid- forming synthetic peptides from a yeast prion protein sequence, discovered in Eisenberg's laboratory, provide a key system for determining the elusive structure and specific interaction properties of hyperstable cross-beta filaments. Single microcrystal electron diffraction data extending to approximately 0.5 Angstrom units resolution will be correlated with X-ray powder diffraction data to determine the atomic structure of the tightly-paired, water-excluding cross-beta peptide chains in these crystals. Relating the cross-beta atomic architecture to the fibrous ultrastructure of the microcrystals by electron tomography may indicate how the polymorphic lateral accretion of pathological amyloid protofilaments is self-limited. Image analysis of amyloid fibers from three different strains of the yeast [PSI] prion, assembled from nanoprobe or GFP labeled recombinant Sup 35p constructs, should establish if each prion strain propagates by imposing its distinctive insoluble cross- beta structure on the common soluble precursor protein. Determination of the structure of the Congo red complex with the TMV protein stacked-disk aggregate by electron and X-ray fiber diffraction will identify the conformational changes responsible for the amyloid-like stability of the aggregate and define specific binding interactions, which will be valuable for design of drugs to inhibit pathological amyloid assemblies. Development of novel methods of electron microcrystallography, fiber diffraction and image analysis coordinated with X-ray diffraction, spectroscopic and optical imaging methods in our studies on model amyloid structures will help to explain the hyperstability, limited lateral association, autocatalytic assembly and distinctive dye binding properties of the cross-beta protofilaments which form pathological amyloid fibers.